This application is for the continuation of a grant whose long-term objective is the investigation of the mechanism of tumorigenesis by a bacterium, Agrobacterium tumefaciens. A. tumefaciens is the only known case of tumorigenic bacterium. Its study has the advantage that well established techniques of bacterial biochemistry and genetic can be used to elucidate the pathogenic process. Previous research has shown that tumor formation involves the transfer of plasmid DNA from the bacterium to the host cell. The first step is the attachment of Agrobacterium to receptor sites on the host cell surface. Bacterial mutants and biochemical characterization will be used to determine the binding sites on the surface of the bacteria which interact with host cell receptors. Preliminary data suggest that Agrobacterium may produce enzymes which modify the host cell surface to aid attachment and to weaken host cell defenses. Bacterial production of such enzymes will be assayed and the virulence of bacterial mutants unable to produce the enzymes will be examined. Little is known about the steps in tumor formation between bacterial attachment and the emergence of an autonomously growing tumor. In order to elucidate these steps the transformation of normal cells in tissue culture to tumor cells will be achieved and the intermediate steps of tumor formation examined. Nucleic acid hybridization techniques will be used to determine the time and order of bacterial plasmid DNA transfer and to study the progress of the plasmid DNA from its entry into the host to its integration into host nuclear DNA. Avirulent bacterial mutants will be examined to determine whether they complement each other and at what stage in tumor formation they are blocked. It is hoped that these studies will provide insights into the fundamental processes of tumorigenesis.